The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

Riad Efendiev,Zongpei Chen,Rafael T Krmar,Sabine Uhles,Adrian I Katz,Carlos H Pedemonte,Alejandro M Bertorello

doi:10.1074/jbc.m500486200

Abstract

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.

Highlights

Regulation of Naϩ,Kϩ-ATPase activity by hormones within the renal tubule epithelial cells provides control for transepithelial sodium transport and thereby urinary sodium excretion during salt loading or deprivation [1]
Endocytosis of Naϩ,Kϩ-ATPase molecules in response to DA in renal epithelial cells is initiated by adaptor protein-2 binding to the Naϩ,Kϩ-ATPase ␣1-subunit Tyr-537 residue [14] and clathrin recruitment [15]
The fact that 14-3-3 proteins can form dimers [20], thereby creating interactive modules potentially linking serine-phosphorylated residues with diverse signaling molecules (including PI 3-kinase [23,24,25]), prompted us to study the possibility that their association with the Naϩ,Kϩ-ATPase ␣1-subunit may guide the PI 3-kinase regulatory subunit (p85␣) to the proline-rich domain upstream of the Ser-18 phosphorylation site and regulate Naϩ,KϩATPase activity and subunit endocytosis

Summary

Introduction

Regulation of Naϩ,Kϩ-ATPase activity by hormones within the renal tubule epithelial cells provides control for transepithelial sodium transport and thereby urinary sodium excretion during salt loading or deprivation [1]. Clathrin-dependent endocytosis of Na؉,K؉-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the ␣-subunit and the binding of phosphoinositide 3-kinase.

Results

Conclusion